Lactoferrin (LTF) has diverse biological activities and is widely used in functional foods and active additives. Nevertheless, evaluating the proteoform heterogeneity, conformational stability, and activity of LTF remains challenging during its production and storage processes. In this study, we describe the implementation of native mass spectrometry (nMS), glycoproteomics, and an antimicrobial activity assay to assess the quality of LTF. We systematically characterize the purity, glycosylation heterogeneity, conformation, and thermal stability of LTF samples from different sources and transient high-temperature treatments by using nMS and glycoproteomics. Meanwhile, the nMS peak intensity and antimicrobial activity of LTF samples after heat treatment decreased significantly, and the two values were positively correlated. The nMS results provide essential molecular insights into the conformational stability and glycosylation heterogeneity of different LTF samples. Our results underscore the great potential of nMS for LTF quality control and activity evaluation in industrial production.
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